While the entry of leukocytes into the central nervous system (CNS) is fundamental to the pathogenesis of many neuroinflammatory conditions, mechanisms regulating the last and most critical step of this process, transendothelial migration (TEM), remain obscure. But two recent series of findings from separate fields could reveal novel and vital clues to how and where TEM takes place. One, ectopic expression of tight junction (TJ) proteins has been described on circulating leukocytes in patients with relapses of the neuroinflammatory disease multiple sclerosis (MS). Elevated expression of TJ proteins by leukocytes in inflammatory conditions, as well as by stem and malignant cells, might endow these cells with heightened ability to cross tissue barriers ? possibly by enabling transient interactions with endothelial TJ proteins via a ?zipper? mechanism. Two, nano- sized extracellular vesicles (EVs), e.g., exosomes and microvesicles, bearing junctional proteins and shed by endothelial cells have been reported. EVs are elevated in blood during inflammation (including MS), shuttle protein and RNA between cells, and interact with various immune cells to alter their adhesion and migration properties. These collective findings could suggest EVs transfer TJ proteins to leukocytes and serve as the missing links functionally connecting TJ proteins on leukocytes, TEM and neuroinflammation. Specifically, by transferring TJ cargo from endothelial cells to leukocytes in a juxtacrine manner, EVs might enable adherent leukocytes to transiently engage corresponding vascular TJ proteins and, thereby, foster TEM across the highly restrictive endothelium of the blood-brain barrier (BBB). Using state-of-the-art technologies, we will focus on leukocytes displaying claudin 5 (CLN-5) ? a major TJ protein of the BBB ? and test the following hypothesis: Leukocytes exploit CLN-5 and endothelial-derived EVs via novel interactions to extravasate across the BBB during neuroinflammation. Aim 1 will use high-resolution 3D fluorescence imaging and FACS to detect CLN-5+-leukocytes and identify their immunophenotypes and activation states at various times in the blood and different CNS regions of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. Aim 2, will use novel endothelial conditional, eGFP-CLN-5 mice or CLN-5 knockdown mice to determine if leukocytes express CLN-5 endogenously or acquire it exogenously from endothelial cells. Aim 3, will use 3D fluorescence imaging/FACS to analyze transfer of CLN-5 cargo (protein and/or mRNA) from endothelial-derived EVs to leukocytes, and a novel CLN-5 peptidomimetic, Pep5, to establish if this action is CLN-5-dependent. Aim 4, will use a recognized in vitro BBB model and Pep5 to examine the functional role(s) of leukocyte CLN-5 and EVs in promoting TEM. These studies will be the first systematical and methodical approach to address the relationship between leukocyte TJ proteins and EVs, and their concerted role in neuroinflammation. As similar processes might generically operate in other instances of cell extravasation, these studies should reveal common pathogenic mechanisms and highlight new, therapeutic approaches that target EVs. !